title: Cloning and characterization of a biotic-stress-inducible glutathione transferase from Phaseolus vulgaris.
abstract: Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its K m for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g., oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus vulgaris.
authors.affiliation: Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, 11855, Athens, Greece.
authors.forename: Evangelia
authors.initials: E
authors.lastname: Chronopoulou
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Madesis
authors.forename: Athanasios
authors.initials: A
authors.lastname: Tsaftaris
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D019295:Computational Biology; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027805:Phaseolus / Q000187:drug effects / Q000201:enzymology*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013312:Stress, Physiological / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D015262:Xenobiotics / Q000494:pharmacology
chemical_list: D004137:Dinitrochlorobenzene; D011994:Recombinant Proteins; D015262:Xenobiotics; D005982:Glutathione Transferase; D005978:Glutathione